Alternative Genetic Selection in Bacillus anthracis and Francisella tularensis

炭疽芽孢杆菌和土拉弗朗西斯菌的替代遗传选择

• 批准号: 7286467
• 负责人: GEORGE C. STEWART
• 金额: $ 18.69万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-05 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7286467
• 关键词: Antibiotic Resistance; Antibiotics; Arsenites; Bacillus anthracis; Bacteria; Cadmium; Categories; Characteristics; Chromates; Chromosomes; Condition; Engineering; Excision; Facility Construction Funding Category; Francisella tularensis; Genes; Genetic Markers; Goals; Heavy Metals; Herbicides; Mediating; Mercury; Mutate; Numbers; Operon; Organism; Phenotype; Plasmid Cloning Vector; Plasmids; Property; Research Project Grants; Resistance; Site; Streptomyces; System; antimicrobial; base; bialaphos; genetic analysis; genetic selection; mutant; recombinase

DESCRIPTION (provided by applicant): Many of the category A select agent bacteria are naturally sensitive to antibiotics and thus introduction of antibiotic resistance selective markers for genetic studies should be avoided. Non-antibiotic selective markers are limited in their availability and have not been evaluated for use in these bacteria. Markers to be developed should have the characteristics of having the selective phenotype expressed when the gene is present in single copy in the organism, being stable when expressed on multi-copy plasmids, and not exerting polar effects on downstream genes when inserted into a multi-gene operon. Desirable properties also include that the selective agent employed should give a low background when sensitive organisms are plated under selective conditions, and the difference between the MIC concentration of sensitive versus resistant organisms should be large. Furthermore, it would be advantageous to be able to easily and precisely excise the selective marker to create a mutant strain lacking the marker cassette so that the antimicrobial markers can be used iteratively to create multiply mutated strains. In this project, we will evaluate heavy metal resistance markers, including resistance to arsenite, cadmium, chromate, and mercury as well as resistance to the herbicide bialaphos as potential selective markers for genetic analysis of Bacillus anthracis and Francisella tularensis. The minimal sequence required for selection in the two hosts will be determined and mutational studies will be carried out to optimize expression. The use of these markers on multicopy plasmids and single copy insertions will be validated in the two hosts. Construction of a transposon bearing these selective agents will be created and evaluated. Engineering of the selective marker cassettes flanked by loxP sites will be accomplished and use of a Cre recombinase-based system to excise the selection cassette from plasmid or chromosomal targets in these select agent bacteria will be evaluated.

描述（由申请人提供）：许多A类选择剂细菌对抗生素天然敏感，因此应避免引入抗生素耐药性选择标记进行遗传研究。非抗生素选择性标记物的可用性有限，尚未对其在这些细菌中的应用进行评估。待开发的标记物应具有基因在生物体中以单拷贝存在时具有选择性表型表达，在多拷贝质粒上表达时具有稳定性，插入多基因操纵子时不会对下游基因产生极性效应的特点。理想的特性还包括，当敏感生物在选择性条件下电镀时，所使用的选择性试剂应提供低背景，并且敏感生物与耐药生物的MIC浓度之间的差异应该很大。此外，能够容易和精确地切除选择标记以创建缺乏标记盒的突变菌株，以便抗菌标记可以迭代地使用以创建多个突变菌株，这将是有利的。在这个项目中，我们将评估重金属抗性标记，包括对亚砷酸盐、镉、铬酸盐和汞的抗性，以及对除草剂双磷的抗性，作为对炭疽芽孢杆菌和土拉菌的遗传分析的潜在选择性标记。在两个宿主中选择所需的最小序列将被确定，并进行突变研究以优化表达。这些标记在多拷贝质粒和单拷贝插入上的使用将在两个宿主中得到验证。构建一个带有这些选择性试剂的转座子将被创建和评估。将完成loxP位点两侧的选择性标记盒的工程设计，并将评估使用基于Cre重组酶的系统从这些选择剂细菌的质粒或染色体靶标上切除选择盒。